Molecular modeling studies and in vitro screening of dihydrorugosaflavonoid and its derivatives against Mycobacterium tuberculosis

Novel drug regimens against tuberculosis (TB) are urgently needed and may be developed by targeting essential enzymes of Mtb that sustain the pathogenicity of tuberculosis. In the present investigation, series of compounds (5a–f and 6a–f) based on a naturally occurring rugosaflavonoid moiety were evaluated by in silico molecular modeling studies against β-ketoacyl-ACP reductase (MabA) (PDB ID: IUZN) and pantothenate kinase (PanK) (PDB ID: 3AF3). Compounds 5a, 5c, 5d, and 6c, which had docking scores of −8.29, −8.36, −8.17 and −7.39 kcal mol−1, respectively, displayed interactions with MabA that were better than those of isoniazid (−6.81 kcal mol−1). Similarly, compounds 5a, 5c, 5d, and 6c, which had docking scores of −7.55, −7.64, −7.40 and −6.7 kcal mol−1, respectively, displayed interactions with PanK that were comparable to those of isoniazid (−7.64 kcal mol−1). Because of their docking scores, these compounds were screened in vitro against Mycobacterium tuberculosis H37Ra (Mtb) using an XRMA protocol. Among the screened compounds, the dihydrorugosaflavonoid derivatives 5a, 5c, and 5d had IC50 values of 12.93, 8.43 and 11.3 μg mL−1, respectively, and exhibited better inhibitory activity than the parent rugosaflavonoid derivatives. The rugosaflavonoid derivative 6c had an IC50 value of 17.57 μg mL−1. The synthesized compounds also displayed inhibitory activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. The present study will be helpful for the further development of these molecules into antitubercular lead candidates.


Introduction
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which affected approximately 10.4 million people in 2015. 1 The World Health Organization (WHO) introduced the DOTS (Directly Observed Treatment, Short Course) strategy, which has proven successful in effectively achieving treatment rates of higher than 90%.However, the prolonged duration (6-9 months) of the DOTS strategy and spontaneous gene mutations in pathogenic strains have led to resistance to the drugs. 2Hence, the emergence of cases of multi-drug resistance (MDR) and extensive drug resistance (XDR) have increased in recent years.To overcome the problems associated with this pandemic, there is an urgent need to develop effective strategies for treating and controlling TB.One strategy would comprise targeting essential enzymes of Mtb that are relevant to its survival and growth within the host cell.In this context, enzymes that participate in biosynthetic pathways represent attractive targets for the discovery of novel anti-tuberculosis agents.Among these, b-ketoacyl-ACP synthase (KAS) and pantothenate kinase (PanK) are two target enzymes that play important roles in the fatty acid synthesis (FASII) system and the biosynthetic pathway of coenzyme A (CoA), respectively. 3,4b-Ketoacyl-ACP reductase (MabA) comprises a complex group of enzymes responsible for the production of very-long-chain fatty acid derivatives that are the chief precursors of mycolic acids, which are the main constituents of the M. tuberculosis cell wall.6][7][8] Rugosaavonoid is a naturally occurring avonoid isolated by Hu et al. 9 from the ower buds of the plant Rosa rugosa.In our ongoing research to develop simple and cost-effective synthetic methodologies for naturally occurring chromones, we have reported the rst total synthesis of rugosaavonoid and its derivatives and identied their cytotoxic potential towards breast cancer cells. 10Recently, Villaume et al. 11  The results for interactions obtained by these docking studies stimulated us to carry out further in vitro antitubercular screening of derivatives against M. tuberculosis H37Ra (Mtb).

2.1
In silico studies 2.1.1Molecular docking.Molecular docking studies were performed to understand the binding probability of the designed molecules.The docking studies showed that compounds 5a, 5c, 5d and 6c docked with the active pockets of 1UZN (b-ketoacyl-ACP reductase) and 3AF3 (pantothenate kinase) and interacted with the active amino acids.Compounds 5(a-f) and 6(a-f) were surrounded in the active pocket of 1UZN by Gly22, Asn24, Arg25, Gly26, Ile27, Gly28, Asn88, Ala89, Gly90, Ile138, Gly139, Ser140, Pro183, Gly184, Tyr185, Ile186, Thr188, Met190, and Thr191.Compounds 5a, 5c, 5d and 6c displayed non-bonding interactions with Gly139, Ser140, and Ile186.MabA (1UZN) contains the amino acids Ser140, Tyr153, and Lys157, which are linked to form the catalytic triad of MabA.Any mutation in the Ser140 residue results in the complete loss of enzymatic activity.Therefore, the interaction of inhibitors with Ser140 may be considered to be important for inhibition.The amino acid Gly90 has been shown to be involved in the complexation of MabA with its natural cofactor NADPH, whereas any mutation of Gly139 to Ala139 causes complete inactivation of the protein by freezing the catalytic triad into a closed form. 12All the active compounds were in close proximity of the active triad and established interactions with Ser140 and Gly139, as depicted in Fig. 1.
Scheme 1 Synthesis of rugosaflavonoid and its derivatives.The docking scores of 5a, 5c, 5d, and 6c with 1UZN were found to be À8.296,À8.366, À8.175 and À7.398 kcal mol À1 , respectively.The standard compound isoniazid bound in a different manner to Asn24, Arg47 and Val62 with a score of À6.813 kcal mol À1 .Besides, we performed docking studies of quercetin, which is a ligand very well known for its biological potential.We found that quercetin interacted with Arg25, Gly28, Asn88, Gly139, and Gly184 with a docking score of À9.412 kcal mol À1 .
The central beta-sheet and p-loop are highly conserved in all pantothenate kinase enzymes (PanK).Differences can be seen in the surrounding loops and helix.The residues Tyr235 and Asn277 are involved in binding with pantothenate and phosphopantothenate. 13 The active site pocket includes residues such as Hip179, Arg238, Tyr182, and Tyr177. 14During the molecular docking of 5(a-f) and 6(a-f) with PanK (PDB code: 3AF3), compounds 5a, 5c, 5d, and 6c displayed interactions with Ala100, Gly102, Ser104, Hip179 and Arg238 (Fig. 2).
Isoniazid interacted with Lys103, Asp129, Hip179 and Glu201.The docking scores were found to be comparable to that of isoniazid (Table 1).Quercetin interacted with Lys147, Hip179 and Arg238.Another form of PanK (PDB 4BFZ) also displayed similar kinds of interactions with these compounds in the active binding pocket (data not presented).
2.1.2Physicochemical properties and ADME predictions.An in silico prediction of physically signicant and pharmaceutically relevant properties of the molecules was performed using QikProp.The data are presented in Table 2.
2.2 Biological screening 2.2.1 Antitubercular activity.Because of the encouraging results of binding with b-ketoacyl-ACP reductase and pantothenate kinase, the synthesized compounds (5a-5f and 6a-6f) were subsequently screened for their in vitro antitubercular activity against M. tuberculosis H37Ra using an established XTT reduction menadione assay (XRMA).Table 3   IC 50 and MIC values of all the compounds.Among the derivatives that were screened, compounds 5c, 5a, and 5d had IC 50 values of 8.43, 12.93 and 11.3 mg mL À1 , respectively, whereas compound 6c had an IC 50 value in the range of 17.57 mg mL À1 .All the remaining compounds exhibited IC 50 values corresponding to a concentration of >20 mg mL À1 .

summarises the
2.2.2 Basic backbone of avone scaffold and its nomenclature.Flavone moieties contain three rings named as A, B and C. The synthesized compounds mostly have variations in ring B. The screening results for the dihydro derivatives showed variations in percentage inhibition with changes in the substituents on ring B. Amongst the tested compounds, compound 5a, which has a methoxy substituent at the 4 0 position, displayed 50% inhibition at 12.93 mg mL À1 and 90% inhibition at >30 mg mL À1 .The addition of another methoxy group at the 3 0 position of ring B (5b) resulted in a sharp decrease in activity.The replacement of the methoxy group by a halogen atom (5c and 5d) enhanced the inhibition, and 5c and 5d exhibited 50% inhibition at 8.43 mg mL À1 and 11.3 mg mL À1 , respectively.The derivative with a methyl group at the 4 0 position of ring B (5e) displayed moderate activity (Table 3).Rugosaavonoid and its derivatives (6a-f) (double bond at C2]C3) did not exhibit any inhibition except for 6c, which exhibited 90% inhibition at 28.90 mg mL À1 .Villaume et al. 11 also reported the importance of ring B when they screened naturally occurring avones as UGM inhibitors.They observed a total loss of activity aer the removal of ring B. In our case, with variations in ring B we observed declines and increases in inhibitory activity.On comparing the dihydrorugosaavonoid derivatives with the naturally occurring molecule quercetin, we found that our compounds exhibited better inhibition than quercetin.These results indicate that dihydro derivatives of rugosaavonoid are much better antitubercular agents than the avone moiety of rugosaavonoid.Besides, the results of an antibacterial assay showed that 5c and 5d displayed very good antibacterial activity against B. subtilis, with IC 50 values of 6.25 mg mL À1 and 7.24 mg mL À1 , respectively.When tested against S. aureus, 5c, 5d, 6c, 6d and 6f displayed excellent activity, with IC 50 values of 2.77 mg mL À1 , 5.63 mg mL À1 , 6.45 mg mL À1 , 8.16 mg mL À1 and 2.96 mg mL À1 , respectively (Table 4).The compounds with good antibacterial activity were further tested to determine their minimum inhibitory concentration (MIC) using a dose-response curve.Compound 5c exhibited MIC values of 27.34 mg mL À1 and 20.44 mg mL À1 against B. subtilis and S. aureus, respectively.Moreover, 5d and 6c exhibited MIC values of 25.02 mg mL À1 and 27.53 mg mL À1 , respectively, against S. aureus (Table 4).All the compounds (5a-5f and 6a-6f) exhibited IC 50 and MIC values of >30 mg mL À1 against the Gram-negative strains E. coli and P. aeruginosa (Table 4).The results of the in vitro screening of these compounds against Gram-positive and Gram-negative bacteria (<25 is poor, >500 is good).MDCK cells are considered to be a good mimic of the blood-brain barrier; QP log K p , predicted skin permeability; QP log K hsa , prediction of binding to human serum albumin (acceptable range: À1.5 to 1.5); percentage human oral absorption (<25% is low, >80% is high).
demonstrated that the synthesized compounds are more active against the Gram-positive bacteria B. subtilis and S. aureus (Table 4).Finally, these results indicate that the dihydro derivatives of rugosaavonoid are much better antitubercular agents than the avone moiety of rugosaavonoid and are also capable of inhibiting the growth of Gram-positive bacteria.

Conclusions
In the present study, we docked and synthesized dihydrorugosa-avonoid and rugosaavonoid derivatives.The derivatives were evaluated in silico against Mtb b-ketoacyl-ACP reductase (MabA) (PDB ID: 1UZN) and PanK (PDB ID: 3AF3), and their activity was conrmed in vitro in M. tuberculosis H37Ra.The results of this integrated effort explicitly support the efficacy of the dihydror-ugosaavonoid derivatives in inhibiting MTB, which opens new avenues for the further development of these molecules into antitubercular lead candidates.

General methods
All the chemicals used during the reactions were procured from Spectrochem, India. 1 H NMR and 13 C NMR spectra were recorded at room temperature using a Varian spectrometer at 400 MHz and 100 MHz, respectively.Chemical shi values are reported with reference to TMS as an internal standard.The samples were prepared by dissolving the synthesized compounds in DMSO-d 6 , and chemical shis are expressed in d (ppm) and coupling constants (J) in Hz.The abbreviations for the splitting patterns are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, unresolved multiplet; dd, doublet of doublets.Column chromatography was performed on Merck silica gel 60 (230-400 mesh).Analytical thin-layer chromatography was carried out on pre-coated Merck silica gel 60F 254 , and iodine was used as the developing reagent.IR spectra were recorded with a Shimadzu FTIR IR Affinity-1 spectrophotometer.CHNS analysis was performed using an Elementar Vario EL III analyzer.

Molecular docking
The 3D structures of MabA (PDB code: 1UZN) and PanK (PDB code: 3AF3 and 4BFZ) were chosen as ideal target proteins for docking with dihydrorugosaavonoid and rugosaavonoid derivatives.Naturally occurring quercetin and isoniazid were employed as standard ligands.

Protein preparation
The selected PDB les of MabA (PDB Code: 1UZN) and PanK (PDB code: 3AF3 and 4BFZ) used for docking studies were downloaded from the RCSB site (www.rcsb.org)and pre-treated prior to the docking calculations with the help of the Protein Preparation Wizard of the Maestro 11.2 program from Schrödinger.Guidelines for the use of the Protein Preparation Wizard were received from a Maestro online tutorial.These steps were followed: (i) addition of hydrogen atoms to the protein structures; (ii) assignment of bond orders; (iii) removal of crystallographic waters; (iv) regeneration of states; (v) optimization of hydrogen bonds using the PROPKA program from Schrödinger before restrained minimization using the OPLS force eld; and (vi) setting the convergence of heavy atoms at the RMSD of 0.3 Å.

Ligand preparation
The dihydrorugosaavonoid (5a-f) and rugosaavonoid (6a-f) derivatives were selected as ligands.They were sketched using the 2D program (Ligand Preparation Wizard) of Maestro 11.2 and converted into a 3D model using the preset option.LigPrep accomplished several corrections of the ligands and provided low-energy structures with ring conformations.Energy minimization and optimization were performed using the Optimized Potential for Liquid Simulations (OPLS) force eld.Subsequently, one conformation was generated for each ligand using Maestro 11.2 soware from Schrödinger.

Receptor grid generation
Glide molecular docking soware uses one ligand to interact with the X-ray crystal structure of the target protein for evaluation of the receptor grid for the active site.Receptor griddependent molecular docking helps ligands to bind in many possible conformations.Docking grids for both protein structures, namely, 1UZN and 3AF3/4BFZ were created using the receptor grid generation option of Maestro.The grid box was positioned at the center of the cognate ligands of the protein structures complexed with their natural cofactor, and the maximum length of the docked ligands was xed at 20 Å.The scaling factor and partial charge cut-off in van der Waals radius scaling were 0.25 and 1 Å, respectively.For other parameters such as sites, constraints, rotatable groups, and excluded volumes, the default settings of Maestro 11.2 were used.

Glide molecular docking
Aer the generation of the ligand and protein structures and the specication of the grid at the active site of the protein, molecular docking measurements were performed.The Glide molecular docking application uses efficient computational simulation techniques for the evaluation of particular poses and ligand exibility, such as the Glide systematic method, which is a new approach for rapid, precise molecular docking, and the resulting GScore, which is an empirical scoring function that combines several parameters.The GScore is given in kcal mol À1 and includes ligand-protein interaction energies, hydrophobic interactions, hydrogen bonds, internal energies, pi-pi stacking interactions, root mean square deviation (RMSD) and desolvation.The Glide module of the XP visualizer was used to analyze the specic ligand-protein interactions.The dihy-drorugosaavonoids, rugosaavonoids, quercetin and the standard isoniazid were docked with the 3D structures of MabA and PanK with the help of Glide.The best-t compounds for each target were identied by their optimal thermodynamic energy values, types of interactions, bonding potential, and conformations.2).This was prepared as per the method reported by McNulty and Mcleod. 18In brief, 3,5-dihydroxybenzoic acid (6 mmol) in dry acetone was taken.To this mixture, K 2 CO 3 (2.5 mmol) was added under stirring at 40 C for 15 min, and stirring was continued at 60 C for 10 min.Then, dimethyl sulfate (2.2 mmol) was added dropwise over a period of 30 min, and the temperature was increased slowly to 80 C. The reaction mixture was allowed to reux for 6 h.The progress of the reaction was monitored by TLC.Aer completion, the reaction mixture was allowed to cool to room temperature and ltered through a celite bed.The ltered mixture was concentrated to obtain the crude product, which was slowly poured onto crushed ice with constant stirring to obtain a solid.The solid that was obtained was ltered and dried to give methyl 3,5-dimethoxybenzoate (2) in 91% yield.
4.3.2Synthesis of methyl 2-acetyl-3,5-dimethoxybenzoate (3).Methyl 3,5-dimethoxybenzoate (2) (5 mmol) was mixed with acetyl chloride (25 mmol) and carbon disulde (2 mL) under dry N 2 in an ice bath. 19To the reaction mixture AlCl 3 (15 mmol) was added under vigorous stirring.The reaction was allowed to stir for 15 min.The progress of the reaction was monitored by TLC.Aer completion, the reaction mixture was quenched with ice and extracted with ethyl acetate.The organic layer was separated, dried over sodium sulfate and concentrated to give the crude product, which was puried by column chromatography (hexane-ethyl acetate, 70 : 30) to obtain methyl 2acetyl-3,5-dimethoxybenzoate (3) in 52% yield.
4.3.3Synthesis of methyl 2-acetyl-3,5-dihydroxybenzoate (4).Methyl 2-acetyl-3,5-dimethoxybenzoate (3) (4 mmol) was added to chlorobenzene, and AlCl 3 (10 mmol) was added slowly at room temperature. 20The reaction mixture was heated to reux for 1 h.The progress of the reaction was monitored by TLC.Aer completion, the reaction mixture was cooled to room temperature and hydrolysed using 1 N HCl.The reaction mixture was extracted with ethyl acetate.The organic layer was separated, dried over sodium sulfate and concentrated to obtain the crude product, which was puried by column chromatography (hexane-ethyl acetate, 80 : 20) to give clean methyl 2acetyl-3,5-dihydroxybenzoate (4) in 68% yield.

Table 2
Prediction of drug-like properties of the lead molecules by QikProp Maestro 11.2 molecular docking suite a À1 (<25 is poor, >500 is good).Caco-2 cells are a model of the gut-blood barrier; QP log BB, predicted brain/blood partition coefficient; QPP MDCK, predicted apparent MDCK cell permeability in nm s À1 a Ligand CID, PubChem IDs of the lead molecules; Ligand STOCK, Updated library of natural compounds, InterBioScreen (IBS) library; Predicted IC 50 value for blockage of HERG K + channels (acceptable range: above À5.0);QPP Caco, predicted apparent Caco-2 cell permeability in nm s

Table 3
Results for in vitro antitubercular activity of 5a-f and 6a-f against Mtb H37Ra